Nucleic acids encoding targeted therapeutic lysosomal enzyme fusion proteins
The present invention relates in general to therapeutic fusion proteins useful to treat lysosomal storage diseases and methods for treating such diseases. Exemplary therapeutic fusion proteins comprise a lysosomal enzyme, a lysosomal targeting moiety, e.g., an IGF-II peptide, and a spacer peptide. Also provided are compositions and methods for treating Mucopolysaccharidosis Type IIIB (Sanfilippo B Syndrome), comprising a targeted therapeutic fusion protein comprising alpha-N-acetylglucosaminidase (Naglu), a lysosomal targeting moiety, e.g., an IGF-II peptide, and a spacer peptide.
1. A nucleic acid encoding a fusion protein comprising (a) a human α-N-acetylglucosaminidase (Naglu) protein comprising amino acids 24-743 of SEQ ID NO: 1, (b) an IGF-II peptide tag with at least 85% amino acid sequence identity to amino acids 8-67 of SEQ ID NO: 5, and (c) a spacer peptide located between the Naglu protein and the IGF-II peptide tag, wherein the spacer peptide comprises an amino acid sequence selected from the group consisting of
(SEQ ID NO: 36)
GGGGSGGGGSGGGGSGGGGSGGGPS,
(SEQ ID NO: 47)
GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST
GPSGAP,
(SEQ ID NO: 51)
GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP,
(SEQ ID NO: 55)
GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP,
and
(SEQ ID NO: 71)
GGGGAGGGGAGGGGAGGGGAGGGPS.
2. The nucleic acid of claim 1 , wherein the IGF-II peptide tag is an N-terminal tag or a C-terminal tag.
3. The nucleic acid of claim 1 , wherein the IGF-II peptide tag is a C-terminal tag.
4. The nucleic acid of claim 1 , wherein the IGF-II peptide tag comprises amino acids 8-67 of SEQ ID NO: 5.
5. The nucleic acid of claim 1 , wherein the IGF-II peptide tag comprises amino acids 8-67 of SEQ ID NO: 5 except for a mutation at the amino acid position corresponding to position 37 of SEQ ID NO: 5.
6. The nucleic acid of claim 5 , wherein the mutation is a substitution of alanine for arginine at position 37 of SEQ ID NO: 5.
7. The nucleic acid of claim 1 , wherein the Naglu protein comprises amino acids 1-743 of SEQ ID NO: 1, and the IGF-II peptide tag comprises (a) amino acids 8-67 of SEQ ID NO: 5 or (b) amino acids 8-67 of SEQ ID NO: 5 except for a mutation at the amino acid position corresponding to position 37 of SEQ ID NO: 5.
8. A cell containing the nucleic acid of any one of claims 1 or 4 - 7 .
9. A method of producing a fusion protein, comprising culturing a cell containing the nucleic acid of any one of claims 1 or 4 - 7 under conditions that permit expression of said nucleic acid to produce the fusion protein encoded by said nucleic acid.
10. A nucleic acid encoding a fusion protein comprising (a) a human α-N-acetylglucosaminidase (Naglu) protein comprising the amino acid sequence of SEQ ID NO: 1, (b) an IGF-II peptide tag comprising amino acids 8-67 of SEQ ID NO: 5, and (c) a spacer peptide located between the Naglu protein and the IGF-II peptide tag, wherein the spacer peptide comprises an amino acid sequence selected from the group consisting of
(SEQ ID NO: 36)
GGGGSGGGGSGGGGSGGGGSGGGPS,
(SEQ ID NO: 47)
GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTG
PSGAP,
(SEQ ID NO: 51)
GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP,
(SEQ ID NO: 55)
GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP,
and
(SEQ ID NO: 71)
GGGGAGGGGAGGGGAGGGGAGGGPS.
11. A nucleic acid encoding a fusion protein comprising (a) a human α-N-acetylglucosaminidase (Naglu) protein comprising the amino acid sequence of SEQ ID NO: 1, (b) an IGF-II peptide tag comprising amino acids 8-67 of SEQ ID NO: 5, except for a mutation within the region corresponding to amino acid positions 30-40 of SEQ ID NO: 5 such that the mutation abolishes at least one furin protease cleavage site in the IGF-II peptide tag, and (c) a spacer peptide located between the Naglu protein and the IGF-II peptide tag, wherein the spacer peptide comprises an amino acid sequence selected from the group consisting of
(SEQ ID NO: 36)
GGGGSGGGGSGGGGSGGGGSGGGPS,
(SEQ ID NO: 47)
GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTG
PSGAP,
(SEQ ID NO: 51)
GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP,
(SEQ ID NO: 55)
GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP,
and
(SEQ ID NO: 71)
GGGGAGGGGAGGGGAGGGGAGGGPS.
12. The nucleic acid of claim 11 , wherein the IGF-II peptide tag comprises amino acids 8-67 of SEQ ID NO: 5 except for a mutation at the amino acid position corresponding to position 37 of SEQ ID NO: 5.
13. The nucleic acid of claim 7 , wherein the mutation is a substitution of alanine for arginine at position 37 of SEQ ID NO: 5.
14. The nucleic acid of claim 12 , wherein the mutation is a substitution of alanine for arginine at position 37 of SEQ ID NO: 5.
15. The nucleic acid of claim 11 , wherein the IGF-II peptide tag comprises amino acids 8-67 of SEQ ID NO: 5 except for a mutation that consists of a deletion of amino acids 30-40 of SEQ ID NO: 5.
16. A cell containing the nucleic acid of claim 11 .
17. A method of producing a fusion protein, comprising culturing a cell containing the nucleic acid of claim 11 under conditions that permit expression of said nucleic acid to produce the fusion protein encoded by said nucleic acid.